Method for the enzymatic hydrolysis of cholesterol esters

ABSTRACT

A totally enzymatic method for the hydrolysis of cholesterol esters using a lipase having cholesterol esterase activity and a protease and a method for quantitative determination of total cholesterol in compositions containing both free and esterified cholesterol comprising enzymatically hydrolyzing the cholesterol esters with a lipase having cholesterol esterase activity and a protease and determining total cholesterol by gas-liquid chromatography or some other suitable technique are described.

States [4 1 Mar. 4, 1975 METHOD FOR THE ENZYMATIC HYDROLYSIS OFCHOLESTEROL ESTERS [75] Inventors: Charles T. Goodhue; Hugh A.

1 Risley, both of Rochester, NY.

[73] Assignee: Eastman Kodak Company,

Rochester, NY.

22 Filed: Mar. 25, 1974 21 Appl. No.: 454,659

[52'] US. Cl. 195/1035 R, 195/62 [51] Int. Cl. G0l n 31/14 [58] Field ofSearch 195/1035 R, 4, 30

[56] References Cited FOREIGN PATENTS OR APPLICATIONS 2,224,132 11/1973Germany OTHER PUBLICATIONS J. Hyun et al., The J. of Biol. Chem, 244,No. 7, pp

Primary Examiner-Alvin E. Tanenholtz Assistant Examiner-C. A. FanAttorney, Agent, or Firm-A. L. Girard [57] ABSTRACT A totally enzymaticmethod for the hydrolysis of cholesterol esters using a lipase havingcholesterol esterase activity and a protease and a method forquantitative determination of total cholesterol in compositionscontaining both free and esterified cholesterol comprising enzymaticallyhydrolyzing the cholesterol esters with a lipase having cholesterolesterase activity and a protease and determining total cholesterol bygas-liquid chromatography or :some other suitable technique aredescribed.

10 Claims, 1 Drawing Figure 1 METHOD FOR THE ENZYMATIC HYDROLYSIS OFCHOLESTEROL ESTERS FIELD OF THE INVENTION This invention relates to anenzymatic method for hydrolyzing cholesterol esters in complex aqueoussolutions which may contain both free and esterified cholesterol, forexample, blood serum and in particular to a process comprisingenzymatically hydrolyzing cholesterol esters using a lipase havingcholesterol esterase activity and a protease.

BACKGROUND OF THE INVENTION The most common clinical estimations ofcholesterol in blood serum are for total cholesterol. This value is ameasure of cholesterol and cholesterol esters present in the serum andanything else such as cholesterol precursors that respondindiscriminately to the usual tests which are based on reactionsinvolving free cholesterol and require prior conversion of cholesterolesters to *free" cholesterol.

In a well-known conventional procedure, serum is extracted with anorganic solvent, the extract is saponified with alcoholic KOH and theliberated cholesterol is isolated and assayed. These methods require thehandling of corrosive chemicals and are tedious, timeconsuming, and notreadily automated.

German, Offenlegungsschrift No. 2,246,695 published Mar. 29, 1973,describes an enzymatic assay for free cholesterol using a cholesteroloxidase, however, this technique requires hydrolysis of the blood serumcholesterol esters using the cumbersome techniques of the prior artprior to application of the enzymatic assay.

G. Bucolo, and H. David, Clin. Chem., 19 476 (1973) describe alipase-protease system for hydrolyzing serum triglycerides; however, itis specifically stated that cholesterol esters are not hydrolyzed inthis system.

OBJECTS OF THE INVENTION It is therefore an object of the presentinvention to provide a simplified and improved technique for hydrolyzingcholesterol esters.

It is another object of the present invention to provide a reproducibleenzymatic process for the quantitative hydrolysis of cholesterol estersin blood serum,

useful as an initial step in the quantitative determination of totalcholesterol in blood serum.

SUMMARY OF THE INVENTION DETAILED DESCRIPTION OF THE INVENTION Accordingto a preferred embodiment of the present invention, hydrolysis ofcholesterol esters in complex aqueous solutions (referred to hereinaftergenerally as blood serum) is achieved by treating the blood serum with amixture comprising per ml of serum from about 20 to about 50 mg 'of alipase having cholesterol ester ase activity and from about 5 to aboutmg of a protease at a temperature of from about 25 to about C and a pHof from about 6.5 to about 9.5 for about 5 to about 15 minutes,preferably with agitation and in an inert atmosphere. When the foregoingconcentrations of lipase and protease are utilized, the lipase shouldcontainat least about 30 international units per mg and the protease atleast about 10 units per mg. One unit of lipase is defined as the amountof the enzyme which will liberate 1 micro mole of fatty acid in a giventime at a given pH and temperature using a substrate containingesterified fatty acid. For the preferred lipase materials describedhereinbelow the conditions are 1 minute at pH 7 and 37C with olive oilas substrate.

One unit of protease will hydrolyze casein to produce color equivalentto 1 micro mole (181 ug) tyrosine per minute at pH 7.5 and a temperatureof 37C. (Color per Folin-Ciocalteu Reagent). It should, of course, beclear that as the level of enzyme activity per unit by weight ofpreparation increases or decreases, so also will the quantity of'enzymepreparation added vary. Most preferably, the ratio of lipase to proteaseon an activity basis should range from about 3 to about 10 and at leastabout I000 units of lipase should be used per ml of serum. Relativelipase activity to esteraseactivity is usually about 10 to about 50.

Generally speaking and in accordance with illustrative embodiments ofour invention, we contact an aqueous medium containing the cholesterolester, preferably blood serum, which contains both esterified and freecholesterol, with a mixture of a lipase which demonstrates cholesterolesterase activity as defined in Example 3 and a protease. The lipasemaybe of plant or animal origin, but we prefer and find best a microbiallipase such as the lipase from'Cana'ida cylindracca. LipasesfromChronzobacterium viscosum, variant paralipolyticum, crude or purified,the lipase from Rhizopus delemar, purified, for example as noted inFukumoto et al, J. Gen. Appli. Microbial, i0, 257-265 (1964), andlipases having similar activity, which are those described in theaforementioned Bucolo and Davis publication, do not demonstrate therequired cholesterol esterase activity. Specifically preferredcommercial lipase preparations include wheat germ lipase supplied byMiles Laboratories of Elkhart, Ind.,

' Lipase 3000 supplied by Wilson Laboratories, Chicago,

Ill., Steapsin supplied by Sigma. Chemical Co., St. Louis, Mo., (both ofthe last two enzymes are pancreatic enzymes) and Lipase M (from Candidacylindracca) supplied by Enzyme Development Corporation, New York, NY.Screening of lipases for this purpose to determine their cholesterolesterase activity may be accomplished using the technique described inExample (3) below. Using this technique, any lipase which demonstrates va cholesterol esterase activity which releases above about 25 mg%cholesterol in the screening procedure of Example 3 should be considereduseful in the successful practice of the present invention.

Proteases in general may be used in the successful practice of thisinvention. These include by way of example, chymotrypsin, Strepromycesgriseus protease (commercially available under the registered trademarkPronase), Aspergillus oryzae protease, Bacillus subtilis protease,elastas, papain and bromelain. Mixtures of such enzymes, of course, mayalso be emtein containing solution the most notable and important ofwhich is, of course, blood serum. In the case where a simple nonprotenaceous solution is being assayed, it is possible, as demonstratedin Example 3 below, to obtain ester hydrolysis using only the lipase. Asalso demonstrated by the Examples, however, in a protein containingsolution such as serum, the presence of the protease is essential touseful results.

As will be further elaborated below, the free cholesterol liberatedby'the action of the foregoing enzyme mixture may be assayed in a numberof ways. According to a preferred embodiment of the instant invention,this assay is performed using gas-liquid chromatography.

According to a preferred embodiment of this free cholesterol assaytechnique, an aliquot generally from about 0.5 to about 5 ml of thehydrolyzed aqueous composition to be tested, hereinafter blood serum, ismixed with from about 0.5 to about 2 ml of heptane or some othersuitable organic solvent, for example, isooctane, containing from about25 mg to about 50 mg weight percent octacosane or some other organicsuitable for use as an internal standard. The heptane may, of course, bereplaced with any other solvents suitable for gas-liquid chromatography,for example, isooctane. The solvent mixture is then extracted with wateraccording to conventional techniques, preferably using from about 3 toabout ml of water per ml of solvent solution. The water extractedsolvent solution is then reacted with a silylating agent, for example,(N,O-bis(- trimethylsilyl) trifluoroacetanide with 1%trimethylchlorosilane or a mixture of equal volumes oftrimethylchlorosilane and 1,1,1,3,3,3,-hexamethyldisilazane for a periodof from about 2 to about minutes. The silylated solvent solution is thenpassed through a conventional gas-liquid chromatograph to determine thetotal cholesterol in the sample under examination. This method fordetermining cholesterol concentration is an adaptation of the techniquedescribed in detail in J. L. Driscoll, D. Aubuchon, M. Descoteaux and R.F. Martin, Anal. Chem., 43, 1196 (1971).

One of the most significant advantages of the instant enzymatichydrolysis technique involves the requirement for dilution of bloodserum for hydrolysis and assay. Surprisingly, using the techniquesdescribed herein, undiluted serum is hydrolyzed as rapidly and readilyas diluted serum. This is quite surprising in view of the uniformrequirements for serum dilution described in the prior art.

The following examples serve to illustrate particular embodiments of thepresent invention.

Validate, a reconstituted serum standard produced by the Warner-LambertCompany, was used in the examples below. The total cholesterol contentof Validate" (lot 2560121) was checked by saponifying an aliquotaccording to the method of Driscoll et a1, and analyzing the heptaneextract by both glc and the Liebermann-Burchard method. Values of 160and 162 mg% respectively were obtained. These are well within the rangeof values quoted by the supplier 148-192 mg%).

HYDROLYSIS OF CHOLESTERO ESTER IN SERUM Y A mixture of 1 ml Validate (aserum cholesterol standard sold by Warner-Lambert and containing 148-192mg% cholesterol), 40 mg Lipase M, 40 mg papain, 8 mg a-chymotrypsin, and0.1 M tris buffer to 3 ml total volume (pH 7.2) is incubated in a 25 mlflask under nitrogen at 50C and 250 rpm for 10 min.

The hydrolysis is performed in an atmosphere of nitrogen in order tominimize artifacts introduced by autooxidation of cholesterol and itsesters; of course, proper correction for such autooxidation factors willpermit hydrolysis to be performed in a normal atmosphere.

EXAMPLE 1 EXAMPLE 2 QUANTITATIVE ESTIMATION OF TOTAL CHOLESTEROL BYGAS-LIQUID CHROMATOGRAPHY (GLC) One ml samples of serum or reconstitutedserum standards (Validate, Warner-Lambert) containing up to 150 mg%cholesterol are mixed with 5 ml ethanol and shaken 3 minutes with 1 mlheptane containing 50 mg7c octacosane. Five ml of water is added and themixture is shaken again for 3 minutes. When the layers separate, equalportions of the heptane layer and N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane are mixed. After 5minutes reaction 1 ul samples are injected into a Hewlett Packard F andM 810 chromatograph with a single stainless steel column (l/8 inch X 4feet) packed with 3% SE30 on 0v 1. Gas flow rate 20 ml/min, oventemperature 250C, injection pop 260C, flame detector 265C, range 10attenuation X l, chart speed k inch/min. Octacosane retention time isabout 1 min. Cholesterol retention time is about 2% min. Runs arecomplete in about 4 min. Under these conditions the amount ofcholesterol in the sample is proportional to the peak height ratios ofcholesterol to octacosane. This method is adapted from Driscoll et a1referred to above.

EXAMPLE 3 SCREENING OF LIPASES FOR CHOLESTEROL ESTERASE ACTIVITY Testswere conducted with cholesteryl linoleate as the substrate because it isthe major ester component of human serum and because it gives relativelystable emulsions compared to saturated esters such as the palmitate.

A solution of 200 mg redistilled cholesteryl linoleate in 5 ml ethylether was mixed with rapid stirring into ml boiling water containing 430mg sodium cholate. Five ml of this suspension was added to a solution of50 mg of lipase preparation in 5 ml 0.1 M phosphate. pH 7.0. Thismixture was incubated 2 hours at 37C, 400 rpm under N Cholesterol estersremaining after this treatment were determined by the hydroxylaminemethod of J. Vonhoeffmayr and R. Fried, Z. Klin. Chem. u Klin.,Biochem., 8, 134 1970) which involves quantitative conversion of estersto hydroxamic acids. The results are shown in Table l.

Table l Hydroxylamine Assay with Cholesteryl Linoleate Suspension All ofthe enzymes show esterase activity. However, Lipase M is preferredbecause of its significantly greater esterase activity and also becauseit is a relatively inexpensive commercial enzyme. As purchased, thepreparation is about 80% lactose, so on a protein basis its activity isabout five times its activity on a weight basis.

EXAMPLE 4 ACTIVITY OF LIPASE WITH SERUM CHOLESTEROL ESTERS Mixturescontaining 40 mg. of lipase in 1 ml Validate" were incubated min. at 50Cunder nitrogen in ml flasks at 250 rpm. The mixture was extracted andcholesterol was estimated as in Example 2. The en zymes tested and theresults are given in Table II.

Table II Esterase Activity with Serum as Substrate Enzyme Cholesterol(mg /r) None 23 Lipase 3000 26 Lipase M 36 Lipase M and lipase 3000while exhibiting considerable esterase activity on cholesteryl linoleateemulsions show very little activity with serum esters.

EXAMPLE 5 ESTERASE ACTIVITY OF LIPASE-PROTEASE COMBINATIONS ON SERUMCHOLESTEROL ESTERS Combinations of Lipase M and various proteases weretested in the same manner as in Example 4. Proteases, except fora-chymotrypsin, were added directly to serum at 40 mg per ml;a-chymotrypsin was added at 8 mg per ml. The amount of cholesterol (mg%)released by each combination is shown in Table III.

Table III It is seen that in the presence of proteases such asa-chymotrypsin or papain the cholesterol esterase activity of Lipase Mwas enhanced nearly four fold. The proteases themselves may have aslight esterase activity but their major effect probably is to increasethe availability of cholesterol esters to the lipase by breaking upester-lipoprotein complexes in serum. Most cholesterol esters in serumare bound to lipoproteins.

Thus, for Lipase M to be optimally effective on serum cholesterolesters, a protease must also be present.

EXAMPLE 6 A series of commercially prepared proteases were tested fortheir ability to enhance esterase activity of Lipase M in serum.Protease was added directly to serum in the amounts shown in Table IV.Lipase M concentration was 40 mg per ml serum. Otherwise assay andconditions are the same as in Example 4. The results are contained inTable IV.

Table IV Amount added Cholesterol Protease (mg/ml) (mg /E l. Aspergillusoryzae 20 I I8 (Sigma Type II) 2. Streplomycex griseus 5 I47 (Sigma TypeVI) 3. Bacillus subrilix 5 I40 (Sigma Type VIII) I0 I I8 20 I35 4.Bromelain 5 I I4 (Sigma Grade II) I0 I27 20 I32 5. Protease 30 20 80(Rohm and Haas) 40 I27 80 l6l 6. Pronase (Calbiochem. Grade A) 5 I33 7.Subtilisin BPN (Sigma Type VII) 5 87 8. Protease, bacterial (Calbiochem.Grade B) I0 I42 9. Lipase M 36 All of the proteases tested appear toenhance the activity ofLipase M somewhat more than a-chymotrypsin andpapain. However, it is difficult: to compare the activities of theseproteases on the basis of units given by suppliers since severaldifferent assays are used. In general, the enzymes judged less pure wereused in the higher amounts.

The effect of pH value on the esterase activity was tested withdifferent buffers and enzymes as described in Examples 7 and 8 below.

EXAMPLE 7 Four different buffers were tested in a system containingthree enzymes. Each sample consists of I ml Validate," 40 mg Lipase M.40 mg papain, 8 mg a-chymotrypsin and 0.I M buffer to 3 ml total volume.The mixtures were incubated and tested as in Example 4. The results aredepicted in FIG. I.

The measurement of pH optimum in this assay with serum as a substratemay be somewhat ambiguous because two enzymes (lipase and protease) arenecessary. It is possible that the pH optimum of each enzyme may notcoincide. In this study tris buffer (tris(hydroxymethyl)aminomethane) atpH 7.2 was superior.

EXAMPLE 8 Studies with tris buffer were conducted on combinations ofLipase M with the series of proteases described in Example 6. The amountof each protease used was that which gave the best result in theexperiment shown in Table IV. Three pH values between 7 and 9 weretested. The assay was the same as described in Example Most of theseproteases show an optimum within the three values tested. The bestresults were obtained with 6 except that time was reduced to 5 minutesso that it bromelain at pH 8.1, Calbiochem bacterial protease at pHvalues from 8 to 9, A. oryzae protease at pH 8.1 and a-chymotrypsin atpH 7.2.

EXAMPLE 9 Table VI Hydrolysis of Cholesterol Esters in Undiluted Serumwith Lipase M and Various Proteases No Additions Trizma Base" TrisBuffer pH 7.2" Amt Cholesterol Cholesterol Cholesterol Protease (mg) pHRange (mg%) pH Range (mg%) pH Range (mg%) 1. B. subrilis 5 7.10-6.89 1048.61-7.85 84 6.98-6.64 208" 2. Protease (Calbiochem) 5 7.03-6.52 1168.52-7.71 100 7.01-6.82 206 3. A. oryzae 80 6.90-6.27 90 7.60-7.25 1036.72-6.70 174 4. Bromelain 6.78-6.56 119 8.25-7.93 52 6.82-6.45 165 5.a-Chymotrypsin 8 7.08-6.93 91 8.65-8.19 51 7.00-6.94 162 6. Pronase 56.82-6.66 134 8.72-7.99 64 7.01-6.89 160 7. S. grr'seus 5 6.88-6.48 968.60-7.94 63 6.89-6.68 119 "Trizma Base" (Sigma) powder was addeddirectly to serum. lris-HCI buffer, pH 7.2 freeze dried, then added aspowder directly to serum.

pH value at start ofincubation and pH value at end.

Some values higher than 160 mg% occasionally obtained from same lotofValidate" calibrated at 160 mg7r.

would be expected to find cholesterol values well below 160 mg%, themaximum available in the Va1idate substrate.

Each incubation mixture contained 0.5 ml Validate, 0.5 ml 0.2M trisbuffer at pH shown, mg 1ipase M and the protease in the amounts shown.lncubation was for 5 minutes under N at C, 250 rpm. Samples wereanalyzed by glc as in Example 2. The results are indicated in Table V.

Although according to a preferred embodiment of the present inventionquantitative estimation of total cholesterol is achieved usinggas-liquid chromatography, any ofthe well known conventional techniquesfor the analysis of total free cholesterol (after cholesterol esterhydrolysis has been achieved) may be used. These include the Pearson,Stern and McGarack, Carr and Drecker, and Zak methods described at pages355-361 of Fundamentals of Clinical Chemistry, Tl- ETZ, N. W., W. B.SAUNDERS CO. (1970) as well as the well known Lieberman-Burchardtechnique and the cholesterol oxidase method described in GermanOffenlegungschrifft No. 2,246,695 referred to hereinabove which isincorporated herein by reference insofar as it describes a technique forquantitatively determining total free cholesterol by treating acholesterol solution with cholesterol oxidase and measuring the quantityof one or more of the products of this oxidation. Furthermore,hydrolysis as described herein may be used as an integral part of asingle solution assay using cholesterol oxidase as is described inconcurrently filed US. Pat. application Ser. No. 454,622 filed Mar. 25,1974. in the names of Goodhue. Risley and Snoke entitled Method andComposition for Blood Serum Cholesterol Analysis," which is incorporatedherein by reference insofar as it describes another useful applicationofthe novel hydrolysis described herein.

While the invention has been described in detail with particularreference to preferred embodiments thereof, it will be understood thatvariations and modifications can be effected within the spirit and scopeof the inventron.

What we claim is:

1. In a process of assaying an aqueous proteinaceous liquid containingcholesterol esters for total cholesterol content in which said ester ishydrolyzed to liberate all of said cholesterol followed by determiningthe amount of cholesterol, the improvement comprising effecting saidhydrolysis by treating said aqueous proteinaceous liquid with both alipase having cholesterol esterase activity and a protease.

2. The improved method of claim 1 wherein said lipase having cholesterolesterase activity releases at least 25 mg% cholesterol (in 2 hours at37C under nitrogen) when 50 mg of a preparation of said lipase in 5 ml0.1 M phosphate buffer, pH 7.0, is used to treat a dispersion of'cholesteryl linoleate prepared by dispersing 200 mg cholesteryllinoleate in 5 ml of ethyl ether and 100 ml boiling water containing 430mg of sodium cholate.

3. The improved method of claim 1 wherein said aqueous liquid is serumand said treating step is accomplished with a mixture comprising fromabout 600 to about 1500 units of said lipase having cholesterol ester-10 ase activity and from about 50 to about 500 units of said proteaseper ml of serum.

4. The method of clain 1 wherein said treatment is carried out at atemperature of between about 25 and 55C and at a pH of between about 6.5and about 9.5.

5. The improved method of claim 1 wherein said lipase having esteraseactivity is a microbial lipase.

6. The improved method of claim 5 wherein said microbial lipase is thelipase from Candida cylindracca.

7. The improved method of claim 1 wherein said lipase is selected fromthe group consisting of wheat germ lipase, pancreatic lipases and thelipase from Candida cylindracca.

8. The improved method of claim 1 wherein said protease is selected fromthe group consisting of a-chymotrypsin, papain, bromelain, Bacillussubll'lis protease, Aspergillus oryzae protease, Streptomyces griseusprotease and mixtures thereof.

9. The improved method of claim 8 wherein said lipase is selected fromthe group consisting of wheat germ lipase, pancreatic lipases and thelipase from Candida cylindracca.

10. The method of claim 9 wherein said treatment is carried out at atemperature of between about 25 and 55C and at a pH of between about 6.5and about 9.5. l= l

1. IN A PROCESS OF ASSAYING AN AQUEOUS PROTEINACEOUS LIQUID CONTAININGCHOLESTEROL ESTERS FOR TOTAL CHOLESTEROL CONTENT IN WHICH SAID ESTER ISHYDROLYZED TO LIBERATE ALL OF SAID CHOLESTER OL FOLLOWED BY DETERMININGTHE AMOUNT OF CHOLESTEROL, THE IMPROVEMENT COMPRISING EFFECTING SAIDHYDROLYSIS BY TREATING SAID AQUEOUS PROTEINACEOUS LIQUID WITH BOTH ALIPASE HAVING CHOLESTEROL ESTRASE ACTIVITY AND A PROTEASE.
 2. Theimproved method of claim 1 wherein said lipase having cholesterolesterase activity releases at least 25 mg% cholesterol (in 2 hours at37*C under nitrogen) when 50 mg of a preparation of said lipase in 5 ml0.1 M phosphate buffer, pH 7.0, is used to treat a dispersion ofcholesteryl linoleate prepared by dispersing 200 mg cholesteryllinoleate in 5 ml of ethyl ether and 100 ml boiling water containing 430mg of sodium cholate.
 3. The improved method of claim 1 wherein saidaqueous liquid is serum and said treating step is accomplished with amixture comprising from about 600 to about 1500 units of said lipasehaving cholesterol esterase activity and from about 50 to about 500units of said protease per ml of serum.
 4. The method of clain 1 whereinsaid treatment is carried out at a temperature of between about 25* and55*C and at a pH of between about 6.5 and about 9.5.
 5. The improvedmethod of claim 1 wherein said lipase having esterase activity is amicrobial lipase.
 6. The improved method of claim 5 wherein saidmicrobial lipase is the lipase from Candida cylindracca.
 7. The improvedmethod of claim 1 wherein said lipase is selected from the groupconsisting of wheat germ lipase, pancreatic lipases and the lipase fromCandida cylindracca.
 8. The improved method of claim 1 wherein saidprotease is selected from the group consisting of Alpha -chymotrypsin,papain, bromelain, Bacillus subtilis protease, Aspergillus oryzaeprotease, Streptomyces griseus protease and mixtures thereof.
 9. Theimproved method of claim 8 wherein said lipase is selected from thegroup consisting of wheat germ lipase, pancreatic lipases and the lipasefrom Candida cylindracca.
 10. The method of claim 9 wherein saidtreatment is Carried out at a temperature of between about 25 and 55*Cand at a pH of between about 6.5 and about 9.5.